hTERT gene expression regulatory gene

ABSTRACT

Disclosed is a novel substance capable of regulating the expression of a telomerase reverse transcriptase gene in a cell of a mammal. A gene capable of regulating the expression of hTERT, comprising a nucleotide sequence depicted in SEQ ID No: 1 or 2. The expression of a telomerase reverse transcriptase gene can be inhibited by inhibiting the expression of the gene. By utilizing this mechanism, the expression of a telomerase reverse transcriptase gene can be regulated.

SEQUENCE LISTING

This patent application incorporates by reference the attached SequenceListing.

TECHNICAL FIELD

The present invention concerns an hTERT expression regulatory gene.

Regarding the enzyme telomerase that is thought to play an extremelyimportant role in the mechanism of carcinogenesis, it is known that thetelomerase reverse transcriptase gene (hTERT) and RNA tape (hTR) are themain regulatory factors for telomerase enzyme expression, and a greatamount of research is being done. However, a direct mechanism linkingtelomerase expression and carcinogenesis has yet to be made clear. Itcan be anticipated that a large number of factors within the genome areinvolved, but the mechanism for telomerase activation is complex, and itis expected that further unknown factors exist.

Additionally, after the year 2000, the role of small RNA called microRNA(miRNA) has been attracting attention, and the elucidation of its rolehas given rise to international competition that is unprecedented in itsintensity. Especially noteworthy is that although miRNA plays animportant role in determining the fate of human cells, the RNA genes andthe miRNA thereof involved in carcinogenesis (particularly telomerase)is not known for human cells.

Additionally, RNA interference (RNAi) has been garnering attention as anefficient technology that blocks the expression of a specific gene. Byusing RNAi technology, researchers can control the expression of genesthat they want to study. Whereby, the cell phenotype in a state wherethis gene is not expressed can be examined, and the gene function can beanalyzed.

At first, it became clear that RNAi is a powerful method for reducingthe expression of a specific gene, by transfecting long double-strandedDNA into invertebrates such as Drosophila and C. elegans. At present,eukaryotic cells including mammalian cells can be analyzed utilizing animproved RNAi knockdown method.

As a conventional RNAi technology, for example, there is that describedin Japanese Unexamined Patent Publication (Kohyo) No. 2002-516062. Inthis RNAi technology, target gene inactivation is performed withdouble-stranded RNA comprising RNA with a sequence homologous to thetargeted gene and its complementary chain. Further, this documentdescribes that it is necessary for the RNA sequence used to be at least50 bases long. Additionally, the experimental examples described in thiscitation use nematodes.

Additionally, as a conventional RNAi technology, there is, for example,that described in Japanese Unexamined Patent Publication (Kohyo) No.2003-529374. This RNAi technology uses siRNA in order to induce RNAi.This document describes (1) structural features of the siRNA, (2) amethod for generating siRNA of approximately 21-23 bases in drosophilaembryo extract having RNAi activity (using Dicer activity therein), (3)chemical synthesis of siRNA, and (4) transfecting and keeping siRNA in acell or individual animal.

Additionally, as a conventional RNAi technology, there is that describedin SAYDA M. ELBASHIR, JENS HARBORTH, WINFRIED LENDECKEL, ABDULLAHYALCIN, KLAUS WEBER, and THOMAS TUSCHL, “Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells”, Nature, 24 May2001, Vol. 411, pp. 494-498. This document recites that by directlytransfecting siRNA that has been cut into short pieces with Dicer intomammalian cells, the RNAi pathway can be made to operate withoutactivation of the interferon pathway.

Additionally, as a conventional RNAi technology, there is, for example,that described in Jurgen Soutschek, Akin Akinc, Birgit Bramlage, KlausCharisse, Rainer Constien, Mary Donoghue, Sayda Elbashir, Anke Geick,Philipp Hadwiger, Jens Harborth, Matthias John, Venkitasamy Kesavan,Gary Lavine, Rajendra K. Pandey, Timothy Racie, Kallanthottathi G.Rajeev, Ingo Rohl, Ivanka Toudjarska, Gang Wang, Silvio Wuschko, DavidBumcrot, Victor Koteliansky, Stefan Limmer, Muthiah Manoharan, andHans-Peter Vornlocher, “Therapeutic silencing of an endogenous gene bysystemic administration of modified siRNAs”, Nature, 11 Nov. 2004, Vol.432, pp. 173-178. This document describes that siRNAs that were linkedto cholesterol sustainedly suppressed the expression of a gene encodingapoB.

Additionally, as a conventional art for suppressing telomerase activity,there is, for example, that described in Japanese Unexamined PatentPublication (Kokai) No. 2002-104995. This document describes a medicinecomprising a combination of a vitamin D receptor agonist such as1,25-dihydroxyvitamin D3 and a retinoid X receptor (RXR) ligand such as9-cis retinoic acid. Additionally, according to this document, thismedicine is a substance that can act directly or indirectly on the humantelomerase reverse transcriptase (hTERT) promoter region, and reduce thedegree of its expression, and is a medicine that is useful as atelomerase activation suppressor and an anti-cancer drug.

Additionally, as a conventional art for suppressing telomerase activity,for example, there is that described in Japanese Unexamined PatentPublication (Kokai) No. 2004-350607. This document describes a doublestranded polynucleotide showing an RNA interference effect against thehuman telomerase reverse transcriptase gene. This document describesthat this double stranded polynucleotide is designed based upon thehuman telomerase reverse transcriptase gene sequence, and it inhibitsthe expression of the human telomerase reverse transcriptase gene.

Additionally, as a conventional art for suppressing telomeraseactivation, there is, for example, that described in the specificationof U.S. Patent Application No. 2003/0099616. This document describes adual specificity tumor killing vector driven by the telomerase promoter.Additionally, this document describes that this expression vector thatoperates due to the telomerase promoter can target tumor cells, and canbe used for RNAi.

However, the conventional art described in the above-mentioned documentshave room for improvement on the following points. First, with theabovementioned conventional art described in Patent Citation 3 andPatent Citation 4, there is room for improvement with regard to thesuppression of telomerase activity. Most of all, since telomerase is alarge molecule of approximately 4.0 kb, human control thereof isdifficult, so there is room for further improvement with regard to theefficiency of telomerase activity suppression and stability.

Second, for the abovementioned conventional art described in PatentCitation 1, Patent Citation 2, Patent Citation 5, Non-Patent Citation 1,and Non-Patent Citation 2, whereas the development of areas having to dowith methods for designing and administering drug candidate substancesthat are useful for anti-cancer suppression is advancing, the discoveryof a raw sample that can be a design source for such drug candidatesubstances has been delayed. That is, since reports of novel factors andmechanisms concerning activation of telomerase are scarce (particularlyactivation of expression of the telomerase reverse transcriptase gene),the raw samples that can be a design source for a drug candidatesubstances are in short supply, so there is room for further improvementwith regard to the degree of freedom of design of drug candidatesubstances that suppress telomerase activity.

DISCLOSURE OF THE INVENTION

The present invention was made in view of the above-mentionedcircumstances, and has the aim of providing a novel substance thatregulates the expression of telomerase reverse transcriptase within amammalian cell.

According to the present invention, an hTERT expression regulatory genecomprising the base sequence shown in SEQ ID No. 1 is provided.

If the expression of this gene comprising the base sequence shown in SEQID No. 1 is inhibited, the expression of telomerase reversetranscriptase is suppressed. Additionally, if this gene comprising thebase sequence shown in SEQ ID No. 1 is overexpressed, the expression oftelomerase reverse transcriptase is suppressed. Therefore, according tothis constitution, the expression of telomerase reverse transcriptasecan be regulated.

Additionally, according to the present invention, products of hTERTexpression regulatory RNA comprising the base sequence shown in SEQ IDNo. 3 and the like are provided.

If the expression of this hTERT expression regulatory RNA comprising thebase sequence shown in SEQ ID No. 3 is suppressed, the expression oftelomerase reverse transcriptase is suppressed. Additionally, if thishTERT expression regulatory RNA comprising the base sequence shown inSEQ ID No. 3 is overexpressed, the expression of telomerase reversetranscriptase is suppressed. Whereby, according to this constitution,the expression of telomerase reverse transcriptase can be regulated.

Further, since the description given above may seem prima facie to beinconsistent, an explanation shall be provided below in order to avoidmisunderstanding. FIG. 27 is a conceptual diagram summarizing the mutualcontrol relationship predicted for the RGM376 gene (SEQ ID No. 1), theRGM249 gene (SEQ ID No. 3), and the hTERT gene. In FIG. 27, + indicatespromotion, whereas − indicates suppression. That is, as can be seen fromthe experimental data from the below-described Embodiment 1 andEmbodiment 2, the RGM 376 gene (SEQ ID No. 1) promotes the expression ofthe hTERT gene when at a normal level of expression, but ifoverexpressed, it suppresses the expression of the hTERT gene. Becauseof this, the expression of the hTERT gene will be suppressed whether theexpression of the RGM376 gene (SEQ ID No. 1) is inhibited, or whether itis overexpressed.

Additionally, as can be seen from the experimental data from thebelow-described Embodiment 1 and Embodiment 2, the RGM249 gene (SEQ IDNo. 3) promotes the expression of the hTERT gene when at a normal levelof expression. On the other hand, it suggests that the RGM249 gene (SEQID No. 3), when overexpressed, promotes the expression of the RGM376gene (SEQ ID No. 1) and makes the RGM376 gene (SEQ ID No. 1) beoverexpressed (data not shown). Therefore, it can be supposed that ifthe RGM249 gene (SEQ ID No. 3) is overexpressed, the expression of thehTERT gene will be suppressed as a result. Therefore, it can be supposedthat the expression of the hTERT gene (SEQ ID No. 3) is suppressedwhether the expression of the RGM249 gene (SEQ ID No. 3) is inhibited,or whether it is overexpressed.

Additionally, according to the present invention, a double stranded RNAis provided that includes a first base sequence with a length of 15bases or longer and 30 bases or shorter, that corresponds to one portionof the abovementioned hTERT expression regulatory RNA, and a second basesequence with a length of 15 bases or longer and 30 bases or shorterthat is complementary to this first base sequence.

This double stranded RNA suppresses the expression of the abovementionedhTERT expression regulatory RNA. Additionally, if the expression of theabovementioned hTERT expression regulatory RNA is suppressed, theexpression of the telomerase reverse transcriptase gene is suppressed.Whereby, according to this constitution, the expression of thetelomerase reverse transcriptase gene can be regulated.

Further, the first base sequence may have a length of 18 or more basesand 25 or less bases. Additionally, the second base sequence may alsohave a length of 18 or more bases and 25 or fewer bases. This is becauseany double-stranded RNA containing a base sequence with a length withinthis range has its function as siRNA or miRNA improved.

Additionally, the present invention also provides single stranded RNAincluding a first base sequence having a length of 15 or more bases and30 or fewer bases, corresponding to one portion of the hTERT expressionregulatory RNA, and a second base sequence having a length of 15 or morebases and 30 or fewer bases disposed in a direction opposite to that ofthe first base sequence, and complementary to the first base sequence.

This single-stranded RNA suppresses the expression of the abovementionedhTERT expression regulatory RNA. Additionally, if the expression of theabovementioned hTERT expression regulatory RNA is suppressed, theexpression of the telomerase reverse transcriptase gene is suppressed.Whereby, according to this constitution, the expression of thetelomerase reverse transcriptase gene can be regulated.

Further, the first base sequence can have a length of 18 or more basesand 25 or fewer bases. Additionally, the second base sequence can alsohave a length of 18 or more bases and 25 or fewer bases. This is becauseany single-stranded RNA containing a base sequence with a length withinthis range has its function as shRNA improved.

Additionally, the present invention also provides RNA that contains aplurality of types of double stranded RNA that are produced by cleaving,with a micro RNA maturation enzyme, double stranded RNA including theabovementioned hTERT expression regulatory RNA and RNA containing a basesequence complementary to that of this hTERT expression regulatory RNA.Further, examples of micro RNA maturation enzymes are enzymes that areinvolved in the maturation of micro RNA, such as Drosha and Dicer.

This RNA, including a plurality of types of double stranded RNA,suppresses the expression of the abovementioned hTERT expressionregulatory RNA. Additionally, if the expression of the abovementionedhTERT expression regulatory RNA is suppressed, the expression oftelomerase reverse transcriptase is also suppressed. Whereby, accordingto this constitution, the expression of telomerase reverse transcriptasecan be regulated.

Additionally, the present invention provides an hTERT expressionregulatory gene comprising the base sequence shown in SEQ ID No. 2.

If the expression of this gene comprising the base sequence shown inthis SEQ ID No. 2 is inhibited, the expression of the telomerase reversetranscriptase gene is suppressed. Whereby, according to thisconstitution, the expression of the telomerase reverse transcriptasegene can be regulated.

Additionally, the present invention provides an hTERT expressionregulatory RNA comprising the base sequence shown in SEQ ID No. 4.

If the expression of this hTERT expression regulatory gene comprisingthe base sequence shown in this SEQ ID No. 4 is suppressed, theexpression of the telomerase reverse transcriptase gene is suppressed.Whereby, according to this constitution, the expression of thetelomerase reverse transcriptase gene can be regulated.

Additionally, the present invention provides a double stranded RNA thatincludes a first base sequence with a length of 15 bases or longer and30 bases or shorter, that corresponds to one portion of theabovementioned hTERT expression regulatory RNA, and a second basesequence with a length of 15 bases or longer and 30 bases or shorterthat is complementary to this first base sequence.

This double stranded RNA suppresses the expression of the abovementionedhTERT expression regulatory RNA. Additionally, if the expression of theabovementioned hTERT expression regulatory RNA is suppressed, theexpression of the telomerase reverse transcriptase gene is suppressed.Whereby, according to this constitution, the expression of thetelomerase reverse transcriptase gene can be regulated.

Further, the first base sequence can have a length of 18 or more basesand 25 or fewer bases. Additionally, the second base sequence can alsohave a length of 18 or more bases and 25 or fewer bases. This is becauseany double stranded RNA containing a base sequence with a length withinthis range has its function as siRNA or miRNA improved.

Additionally, the present invention also provides single stranded RNAincluding a first base sequence having a length of 15 or more bases and30 or fewer bases, corresponding to one portion of the abovementionedhTERT expression regulatory RNA, and a second base sequence having alength of 15 or more bases and 30 or fewer bases disposed in a directionopposite to that of the first base sequence, and complementary to thefirst base sequence.

This single stranded RNA suppresses the expression of the abovementionedhTERT expression regulatory RNA. Additionally, if the expression of theabovementioned hTERT expression regulatory RNA is suppressed, theexpression of the telomerase reverse transcriptase gene is suppressed.Whereby, according to this constitution, the expression of thetelomerase reverse transcriptase gene can be regulated.

Further, the first base sequence can have a length of 18 or more basesand 25 or fewer bases. Additionally, the second base sequence can alsohave a length of 18 or more bases and 25 or fewer bases. This is becauseany single stranded RNA containing a base sequence with a length withinthis range has its function as shRNA improved.

Additionally, the present invention also provides RNA that contains aplurality of types of double stranded RNA that are produced by cleaving,with a micro RNA matureation enzyme, double stranded RNA including theabovementioned hTERT expression regulatory RNA and RNA containing a basesequence complementary to that of this hTERT expression regulatory RNA.Further, examples of micro RNA maturation enzymes are enzymes that areinvolved in the maturation of micro RNA, such as Drosha, Dicer, Ago2,and TRBP.

This RNA including a plurality of types of double stranded RNAsuppresses the expression of the abovementioned hTERT expressionregulatory RNA. Additionally, if the expression of the abovementionedhTERT expression regulatory RNA is suppressed, the expression oftelomerase reverse transcriptase is also suppressed. Whereby, accordingto this constitution, the expression of telomerase reverse transcriptasecan be regulated.

Further, the abovementioned phenomena are all phenomena occurring withinmammalian cells.

Additionally, the abovementioned genes and RNA are not limited to theabovementioned base sequences, but genes and RNA comprising basesequences wherein one or a few of the bases in these base sequences aremissing or replaced, or are added to, also have similar effects.

Additionally, the abovementioned genes and RNA are not limited to theabovementioned base sequences, and genes and RNA comprising basesequences of mammal-derived polynucleotide molecules that hybridizeunder stringent conditions to polynucleotide molecules comprising basesequences that are complementary to the polynucleotide moleculescomprising these base sequences also have similar effects.

Additionally, the abovementioned genes and RNA are one mode of thepresent invention, and the genes and RNA of the present invention may bearbitrary combinations of the constituent elements given above.Additionally, vectors and complexes of the present invention also have asimilar constitution, and have similar effects.

That is, according to the present invention, the expression of thetelomerase reverse transcriptase gene within mammalian cells can beregulated because either one of two novel types of genes that regulatethe expression of the telomerase reverse transcriptase gene, orsubstances obtainable based upon these genes are used.

BRIEF DESCRIPTION OF DRAWINGS

[FIG. 1] Physical map showing the position of gene 1 and gene 2 in thevicinity of band 15.1 on the short arm of human chromosome 10.

[FIG. 2] Conceptual diagram for explaining the procedure for cloninggene 1.

[FIG. 3] Conceptual diagram for explaining the location on humanchromosome 10, and the structure of gene 2.

[FIG. 4] Conceptual diagram for explaining the structure and relativeposition of RNA that is a transcription product of gene 1 and gene 2.

[FIG. 5] Diagram for explaining the sequence of gene 1 and gene 2.

[FIG. 6] Diagram for explaining the sequence of a variant of gene 1.

[FIG. 7] Diagram for explaining the sequence of siRNA designed basedupon gene 1.

[FIG. 8] Diagram for explaining the sequence of siRNA designed basedupon gene 2.

[FIG. 9] Diagram for explaining method for transfecting RNA into a cell.

[FIG. 10] Frame format showing effect of RNAi.

[FIG. 11] Electrophoresis diagram for explaining dicing of two types ofmRNA by Dicer.

[FIG. 12] Electrophoresis diagram showing differentiation specificexpression of RGM376 in liver cancer cells.

[FIG. 13] Electrophoresis diagram and graph for explaining suppressionof telomerase activity due to overexpression of RGM376.

[FIG. 14] Electrophoresis diagram showing expression of hTERT and RGM376in a transformed cell.

[FIG. 15] Electrophoresis diagram showing expression of a gene in anRGM376 siRNA-transfected cell.

[FIG. 16] Electrophoresis diagram and graph summarizing hTERT results ofsuppression due to RGM376 siRNA.

[FIG. 17] Electrophoresis diagram showing differentiation specificexpression of RGM249 in a liver cancer cell.

[FIG. 18] Electrophoresis diagram showing expression of genes due totransfection of RGM249 dsRNA into HMc-Li7.

[FIG. 19] Electrophoresis diagram showing expression of genes due totransfection of RPL22 dsRNA into HM.

[FIG. 20] Electrophoresis diagram showing expression of genes due totransfection of RGM249-specific siRNA into HLF.

[FIG. 21] Graph summarizing rate of suppression of mRNA expression ofeach gene after transfection of RGM249-specific siRNA.

[FIG. 22] Electrophoresis diagram showing expression of RGM376 due totransfection of RGM249 siRNA, and expression of RGM249 due totransfection of RGM376 siRNA.

[FIG. 23] Conceptual diagram for explaining the proposed mechanism fortranslational silencing due to micro RNAs.

[FIG. 24] Conceptual diagram for explaining mechanism wherein shRNA isgenerated from plasmids for generating shRNA, and the expression oftarget gene is inhibited.

[FIG. 25] Electrophoresis diagram showing expression of mRNA of eachgene after transfection with RGM249 shRNA.

[FIG. 26] Graph summarizing rate of suppression of mRNA expression ofeach gene after transfection with RGM249 shRNA.

[FIG. 27] Conceptual diagram summarizing expected mutual controlrelationship between RGM376 gene (SEQ ID No. 1), RGM249 gene (SEQ ID No.3), and hTERT gene.

[FIG. 28] Diagram for explaining sequence of RGM249 shRNA to be designedbased upon gene 2.

[FIG. 29] Diagram for explaining sequence of RGM249 variant shRNA to bedesigned based upon gene 2.

BEST MODE FOR EMBODYING THE INVENTION

Herebelow, a mode for embodying the present invention shall be explainedusing figures. In all of the figures, identical reference numbers shallbe associated with identical constituent elements, and explanationsshall be appropriately omitted.

<Explanation of Telomerase>

The present mode of embodiment concerns telomerase, an enzyme thought toplay an extremely important role in the mechanism of carcinogenesis. Inthe present embodiments, a functional analysis shall be carried out oftwo RNA genes that are closely involved in, and control, the expressionof telomerase reverse transcriptase (hTERT), which is the primarycandidate for carcinogenesis, that is, a cancer antigen.

Telomerase is an enzyme that is needed for the elongation of thetelomere sequences at the ends of chromosomes and the stability ofchromosomes, and is deeply involved in escaping from cell aging, and theinducing of canceration.

Examples of molecules that are necessary for the formation of telomeraseare hTERT (human telomerase reverse transcriptase), hTR (humantelomerase RNA tape: RNA component), TEP1 (telomerase-associated protein1), Dyskerin (DKC1), RPL22, Nola3, La-antigen, Staufen, and H/ACA, andthe activation and generation of telomerase enzymes is controlled bythese molecules.

<Position of the Two Genes on the Chromosome and the Background of TheirDiscovery>

FIG. 1 is a physical map showing the position of gene 1 and gene 2 inthe vicinity of band 15.1 on the short arm of human chromosome 10.

Both of the genes cloned from the short arm of human chromosome 10 bythe present inventor as telomerase controlling genes are mapped onadjacent positions on approximately 1.5 kb on the genome. Further, inthe diagram, on the chromosome shown on the left, the upper direction isthe telomere direction, and the lower direction is the centromeredirection.

Gene1 shows the location of gene 1, which the present inventor namedRGM376. Additionally, Gene2 shows the location of gene 2, which thepresent inventor named RGM249. Further, between Gene1 and Gene2 existDNA markers that are named D10S1728 and D10S1649.

Gene1, D10S1728, D10S1649, and Gene2 all exist in a common region with alength of 1.5 kb in the genome library, names AL355591.3, AL138774.5,J21, H11, and M6.

FIG. 2 is a conceptual diagram for explaining the procedure for cloninggene 1. The present inventor, in order to clone the RGM376 gene (gene1), first performed exon trapping, after having performed the micronuclear fusion method and BAC screening. Next, for the region narroweddown by exon trapping, the identification of an exon that suppressestelomerase activity and the expression of hTERT mRNA was performed byforced expression.

Then, as a result of an examination, ORF analysis, and in vitrotranslation of the base sequence of the identified exon of the RGM376gene, it was strongly suggested that the RGM376 gene (gene 1) is anncRNA that does not synthesize a protein.

Further, the RGM 249 gene (gene2) is a gene that was identified as anexon within a region of length 1.5 kb that is shared by a plurality ofclones obtained during the cloning of the RGM376 gene (gene 1).

This RGM249 gene (gene 2) was also selected out as a gene that isexpressed in cancer cell lines, from 44 exons obtained by exon trappingusing a BAC library of the 10p14-15 region of the short arm of humanchromosome 10, whereon the existence of a telomerase and cancer-relatedcontrol gene was suggested, and cDNA was obtained.

As a result of having performed upon the identified RGM249 gene theanalysis of the base sequence of its exons, and in vitro transcriptionand translation using protein expression vectors, it was stronglysuggested that the RGM249 gene (gene 2) is an ncRNA that does not encodea protein.

<Explanation of Structure and Function of the Two Genes

FIG. 3 is a conceptual diagram for explaining the location upon humanchromosome 10 and the structure of gene 2. The RGM249 gene (gene2)exists at band 15.3 on the short arm of human chromosome 10.Additionally, the length of the RGM249 gene is 249 bp. Further, theRGM249 gene comprises two exons. One exon has a length of 190 bp, whilethe other exon has a length of 59 bp.

The RGM376 gene (gene 1) is also similarly located at band 15.3 on theshort arm of human chromosome 10. Additionally, the length of the RGM376gene is 376 bp.

FIG. 4 is a conceptual diagram for explaining the structure and relativeposition of the RNA that is a transcription product of gene 1 and gene2. FIG. 4( a) shows the putative two-dimensional structure of the RNAthat is a transcription product of the RGM376 gene. Additionally, FIG.4( b) shows the putative two-dimensional structure of the RNA that is atranscription product of the RGM249 gene.

FIG. 4( c) shows the relative position of the RGM376 gene and the RGM249gene on the short arm of human chromosome 10. From the fact that, inthis way, the RGM249 gene is located upstream from the RGM376 gene, amechanism that integrates and controls these genes can be supposed toexist, and the involvement of both genes in the mechanism ofcarcinogenesis is suggested.

One of these RNA genes, the RGM376 gene (gene 1), functions to suppresshTERT due to overexpression, and the other RNA gene, the RGM249 gene(gene 2), functions synchronously with hTERT expression.

More specifically, as shall be described below, there are two of theseRNA genes, one that is expressed in liver cancer and behaves in unisonwith the expression of hTERT (gene 2), and one that is expressed inliver cells, and functions suppressively against cancer cells (gene 1),and they have functions that are mutually conflicting. Additionally, asshall be described below, the former (gene 2), after suppressing thetelomerase gene with dsRNA, causes the cell death of cancer cells, andthe latter (gene 1) acts suppressively against liver cancer, lungcancer, breast cancer, and the like by forced expression.

Additionally, from FIG. 4( a) and FIG. 4( b), there is the possibilitythat by dicing with enzymes such as Drosha and Dicer, miRNA is producedwithin living organisms from this RNA. In actuality, as described below,miRNA cluster genes (gene 1 and gene 2) that have been cloned from theshort arm of human chromosome 10, as telomerase control genes, producemiRNA of 20 mer to 60-70 mer by the action of enzymes such as Drosha andDicer. Experimental data also strongly suggests that in this way, microRNA is produced, and it is involved in control. Further, by designingand constructing small functional RNA such as these miRNA, applicationto highly specific anti-cancer genetic medicine can be expected.

FIG. 5 is a diagram for explaining the sequence of gene 1 and gene 2.FIG. 5( a) shows the base sequence of the RGM376 gene (gene 1). TheRGM376 gene has a length of 376 bases. Additionally, FIG. 5( b) showsthe base sequence of the RGM249 gene (gene 2). The RGM249 gene has alength of 249 bases.

FIG. 5( c) shows the base sequence of the RNA that is the transcriptionproduct of the RGM 376 gene (gene 1). The RNA that is a transcriptionproduct of the RGM376 gene has a length of 376 bases. Additionally, FIG.5( d) shows the base sequence of the RNA that is a transcription productof the RGM249 gene (gene 2). The RNA that is the transcription productof the RGM249 gene has a length of 249 bases.

FIG. 6 is a diagram for explaining the sequence of a variant of gene 1.As the result of transfecting an RGM376 variant wherein the mutation wasinduced by Mutazyme, a clone having a mutation at one base (A) thatcorresponds to the 280th base from the 5′ end (muta 376-3-1-2) showedweak growth suppression and telomerase expression suppression(suppression of mRNA expression and suppression of telomerase activity).

That is, a gene comprising a base sequence wherein one or a plurality ofbases from the base sequence of the RGM376 gene (gene 1) are missing,replaced, or added to, also shows telomerase expression suppression(suppression of mRNA expression and suppression of telomerase activity),similarly to gene 1.

Additionally, it is expected that a gene comprising a base sequence ofmammal-derived DNA molecules that hybridize under stringent conditionsto DNA molecules comprising a base sequence that is complementary to theRGM 376 gene (gene 1), also shows suppression of telomerase expression,similarly to gene 1.

Additionally, regarding the RGM249 gene (gene 2) also, it is expectedthat a gene comprising a base sequence wherein one or a plurality of thebases of the base sequence of gene 2 are missing, replaced, or added to,will also function as an hTERT expression regulatory gene thatsynchronizes with the expression of hTERT, similarly to gene 2.

Further, it is expected that a gene comprising a base sequence ofmammal-derived DNA molecules that hybridize under stringent conditionsto DNA molecules comprising a base sequence that is complementary togene 2, will also function as an hTERT expression regulatory gene thatsynchronizes with the expression of hTERT, similarly to gene 2.

Additionally, regarding the RNA that are transcription products of gene1 and gene 2, RNA that comprises base sequences wherein one or aplurality of bases are missing, replaced, or added to the original RNA,are expected to have a similar function to the original RNA.

Further, regarding RNA that is a transcription product of gene 1 or gene2 also, mammal-derived RNA that hybridizes under stringent conditions toRNA molecules comprising base sequences that are complementary to theseoriginal RNA molecules is expected to have a function similar to theoriginal RNA.

FIG. 7 is a diagram for explaining the sequence of siRNA designed basedupon gene 1. FIG. 7( a), A and B are diagrams that show an example of adouble strand (sense strand and antisense strand) of siRNA designedbased upon the RGM376 gene (gene 1). As described below, thisdouble-stranded siRNA shown in A and B suppresses the expression of theRNA of the RGM376 gene (gene 1).

FIG. 7( b) is a diagram that shows a plurality of examples of sensestrands of siRNA designed by Invitrogen (registered trademark) BLOCK-iTRNAi Designer, manufactured by Invitrogen Corporation, based upon theRGM376 gene (gene 1). Among these sense strands of siRNA, the sensestrand of SEQ ID No. 6 corresponds to FIG. 7( a) A.

More specifically, the design of chemically modified siRNA was carriedout using Stealth (registered trademark) RNAi Designer. FIG. 7( b)lists, among the results of function prediction using Stealth RNAiDesigner, those that have a high predicted value for functioning as ansiRNA. The details of chemical modification by Stealth RNAi have notbeen made publicly available, since they are an industrial secret, butthey are obtainable upon ordering from Invitrogen Corporation.

It is expected that any double stranded siRNA whereof the sense strandis a sequence listed in FIG. 7( b), suppresses the expression of the RNAof the RGM376 gene (gene 1), similarly to the siRNA of FIG. 7( a), A andB.

Further, the double stranded siRNA can be Stealth RNAi, but it is notparticularly restricted, and can have overhangs with a length of twobases on the 3′ end of both the sense strand and the antisense strand.Additionally, these RNA can be not only double stranded siRNA, but alsosingle stranded shRNA wherein a hairpin loop sequence with a length of 4bases or more is disposed in between a sense sequence (corresponding tothe sense strand) and an antisense sequence (corresponding to theantisense strand). Further, there is no particular limit on the lengthof this hairpin loop sequence, but it can be, for example, 8 bases long.This is because it is expected that any of these will suppress theexpression of the RNA of the RGM376 gene (gene 1).

FIG. 7( c) is a diagram showing the location on the entire length of thesequence of the RGM376 gene (gene 1) that corresponds to the sequence ofthe sense strand of FIG. 7( a) A and B. The underlined portionsindicated by the reference letters A and B correspond to the sequencesof the sense strands of A and B.

FIG. 8 is a diagram for explaining the sequence of siRNA that isdesigned based upon gene 2. FIG. 8( a), (1) (the number is circled inthe diagram) and (2) (the number is circled in the diagram) are diagramsthat show an example of a double strand (sense strand and antisensestrand) of siRNA designed based upon the RGM249 gene (gene 2). Asdescribed below, this double stranded siRNA shown in (1) and (2)suppresses the expression of the RNA of the RGM249 gene (gene 2).

FIG. 8( b) is a diagram that shows a plurality of examples of sensestrands of siRNA designed by Invitrogen (registered trademark) BLOCK-iTRNAi Designer, manufactured by Invitrogen Corporation, based upon theRGM249 gene (gene 2). Among the sense strands of these siRNA, the sensestrand of SEQ ID No. 20 corresponds to FIG. 7( a) (1) (the number iscircled in the diagram). Additionally, the sense strand of SEQ ID No. 32corresponds to a sequence (2)′ (indicated in the diagram by a circlednumber), which is a sequence wherein the entire base sequence of theRGM249 gene (gene 2) is shifted one base towards the 5′ end.

Similarly with the case for FIG. 7, in FIG. 8 also, the design ofchemically modified siRNA is carried out using Stealth RNAi Designer.That is, FIG. 8( b) lists, among the results of function predictionusing Stealth RNAi Designer, those that have a high predicted value forfunctioning as an siRNA. The details of chemical modification by StealthRNAi have not been made publicly available, since they are an industrialsecret, but they are obtainable upon ordering from InvitrogenCorporation.

It is expected that any double stranded siRNA whereof the sense strandis a sequence listed in FIG. 8( b), suppresses the expression of the RNAof the RGM249 gene (gene 2), similarly to the siRNA of FIG. 8( a), (1)and (2).

Further, the double stranded siRNA can be Stealth RNAi, but it is notparticularly restricted, and can have overhangs with a length of twobases on the 3′ end of both the sense strand and the antisense strand.Additionally, these RNA can be not only double stranded siRNA, but alsosingle stranded shRNA wherein a hairpin loop sequence with a length of 4bases or more is disposed in between a sense sequence (corresponding tothe sense strand) and an antisense sequence (corresponding to theantisense strand). Further, there is no particular limit on the lengthof this hairpin loop sequence, but it can be, for example, 8 bases long.This is because it is expected that any of these will suppress theexpression of the RNA of the RGM249 gene (gene 2).

FIG. 8( c) is a diagram showing the location on the entire length of thesequence of the RGM249 gene (gene 2) that corresponds to the sequence ofthe sense strand of FIG. 8( a) (1) and (2). The underlined portionsindicated by the reference numbers (1) and (2) correspond to thesequences of the sense strands of (1) and (2).

FIG. 9 is a diagram for explaining the method of transfecting the RNAinto cells. For example, in order to perform the experiments describedin the following embodiments using siRNA and the like including thespecific double stranded RNA designed based upon (1) the double strandedRNA of the RGM249 gene (gene 2), (2) the double stranded RNA of theRPL22 gene, or the above-described RGM249 gene (gene 2), these RNA mustfirst be transfected into mammalian cells. At this time, as atransfection reagent, for example, Lipofectamine 2000 (registeredtrademark), manufactured by Invitrogen Corporation, may be used.

Next, after having cultured under predetermined conditions thesemammalian cells wherein these RNA have been transfected, these RNA areextracted from these mammalian cells. Whereafter, by performing RT-PCRusing these extracted RNA, the changes in the expression of each of thetypes of RNA due to transfection with these RNA can be detected.

FIG. 10 is a schematic diagram showing the effect of RNAi. The siRNAbecomes one portion of an RNA-nuclease complex (RNA-induced silencingcomplex, or RISC) that targets the complementary cellular mRNA. At thistime, the sense strand separates from the antisense strand and is brokendown.

Then, the RNA-nuclease complex that includes the antisense strand ofsiRNA suppresses the expression of the target mRNA within the cell byrecognizing and cleaving target mRNA that is complementary to theantisense strand.

Further, the RNA used in RNAi is not restricted to siRNA havingoverhangs with a length of two bases on the 3′ end of both the sensestrand and the antisense strand, but can also be single stranded shRNAwherein a hairpin loop sequence with a length of 4 bases or more isdisposed in between a sense sequence (corresponding to the sense strand)and an antisense sequence (corresponding to the antisense strand).Further, there is no particular upper limit on the length of thishairpin loop sequence, but it can be, for example, 8 bases long. This isbecause for such shRNA, the hairpin loop sequence thereof is cleaved,and changes to siRNA.

Herebelow, the uses of the two RNA genes of the present mode ofembodiment shall be explained. Since the two RNA genes of the presentmode of embodiment both have functions of regulating the expression ofthe telomerase reverse transcriptase gene (hTERT), they can regulate theexpression of the telomerase reverse transcriptase gene (hTERT) withinmammalian cells.

More specifically, the two RNA genes of the present mode of embodimentare the key to understanding the molecular mechanism of carcinogenesis,and will prove useful for the development of an anti-cancer treatmentusing telomerase suppression (molecular targeting treatment). Whereastelomerase, being a large molecule of 4.0 kb, is especially difficultfor humans to control, said genes are molecules of 400 bp or less, so itcan be thought that regulation of genetic expression would be easier. Inrecent years, much anti-cancer research has been carried out, andalthough the development of fields concerning the administration thereofhas been advancing, the discovery of original samples that are to beadministered has been slow. Since reports about factors that are relatedextremely directly to cancer have been few, the investigation of thisregion, and the fact that this molecule is related to the theme ofcancer eradication, can be thought to be innovative and groundbreaking.

Specifically, regarding the RGM376 gene (gene 1), if the RNA that is atranscription product thereof is overexpressed, the expression of thetelomerase reverse transcriptase gene (hTERT) is suppressed. Thus, bymaking the RNA that is a transcription product of the RGM376 gene(gene 1) be overexpressed, telomerase activation can be suppressed, andas a result, carcinogenesis can be suppressed. Additionally, regardingthe RGM376 gene (gene 1), it can be used as a research tool forinvestigating the molecular mechanism of carcinogenesis.

Additionally, the expression vector constructed by linking the RGM376gene (gene 1) to a vector, when transfected into a cell, suppresses theexpression of the telomerase reverse transcriptase gene (hTERT).Whereby, this expression vector suppresses the activation of telomerase,and as a result, can suppress carcinogenesis.

Therefore, when a medication containing RNA that is a transcriptionproduct of the RGM376 gene (gene 1) or the abovementioned expressionvector is administered to the human body, the expression of thetelomerase reverse transcriptase (hTERT) gene is suppressed. Thus, thismedication suppresses the activation of telomerase, and as a result, issuited for being used as a cancer treating agent that can suppresscarcinogenesis.

Additionally, the various types of RNA that are designed based upon theRGM376 gene (gene 1), such as siRNA and shRNA, suppress the expressionof the telomerase reverse transcriptase (hTERT) gene by suppressing theexpression of the RGM376 gene (gene 1). Additionally, RNA (RNA cocktailor RNA pool) containing a plurality of types of double stranded RNA madeby cleaving with Dicer or the like double stranded RNA including RNAthat is a transcription product of RGM376 (gene 1) and RNA that includesa base sequence that is complementary to this RNA, also suppress theexpression of the telomerase reverse transcriptase (hTERT) gene, bysimilarly suppressing the expression of the RGM376 gene (gene 1).Whereby, these various types of RNA suppress telomerase activity, and asa result, can suppress carcinogenesis.

Additionally, a complex comprised by linking various types of RNA suchas these siRNA and shRNA with a substance that promotes (induces) uptakeinto lipids and cells such as cholesterol, as described in Non-PatentCitation 2, suppresses the expression of target mRNA (RGM376 gene (gene1)) continuously and for a long time within mammalian cells such as inthe human body. Whereby, the various types of complexes such as thisRNA-cholesterol complex, by suppressing the expression of the RGM376gene (gene 1) over a long time, suppress the expression of thetelomerase reverse transcriptase (hTERT) gene. Additionally, if thecomplex is a complex that can be obtained by linking an uptake promoter(inducer) to the various types of RNA, the uptake efficiency into cellswill also improve. Therefore, these various types of RNA suppresstelomerase activity over a long time, and as a result, can suppresscarcinogenesis over a long period of time.

For this reason, medications containing various types of RNA such as theabovementioned siRNA and shRNA, as well as the abovementionedRNA-cholesterol complexes, suppress the expression of the telomerasereverse transcriptase gene when administered to the human body. Whereby,these medication suppress telomerase activity, and as a result, cansuitably be used as cancer treatment agents that can suppresscarcinogenesis. In particular, medications containing the abovementionedRNA-cholesterol complex have the advantage that the intervals betweenadministration to the human body can be made long, because they suppresscarcinogenesis over a long period of time, due to the abovementionedmechanism.

On the other hand, additionally, regarding the RGM249 gene (gene 2),since the expression of RNA that is the transcription product, and theexpression of hTERT (canceration of the cell) synchronize, it can beused as a research tool for investigating the molecular mechanism ofcarcinogenesis. Additionally, an expression vector that is constructedby linking the RGM249 gene (gene 2) to a vector so as to enableexpression, promotes the expression of the telomerase reversetranscription (hTERT) gene when transfected into a cell, and promotestelomerase activity and causes canceration to occur. Therefore, thisexpression vector can also be used as a research tool.

Additionally, the various types of RNA such as siRNA and shRNA that aredesigned based upon the RGM 249 gene (gene 2), suppress the expressionof the telomerase reverse transcriptase (hTERT) gene by suppressing theexpression of the RGM249 gene (gene 2). Additionally, RNA (RNA cocktailor RNA pool) containing a plurality of types of double stranded RNA madeby cleaving with Dicer or the like double stranded RNA including RNAthat is a transcription product of RGM249 (gene 2) and RNA that includesa base sequence that is complementary to this RNA, also suppresses theexpression of the telomerase reverse transcriptase (hTERT) gene, bysimilarly suppressing the expression of the RGM249 gene (gene 2).Therefore, these various types of RNA suppress telomerase activity, andas a result, can suppress carcinogenesis.

Additionally, a complex comprised by linking various types of RNA suchas these siRNA and shRNA with a substance that promotes (induces) uptakeinto lipids and cells such as cholesterol, as described in Non-PatentCitation 2, suppresses the expression of target mRNA (RGM249 gene (gene2)) continuously and for a long time within mammalian cells such as inthe human body. Whereby, the various types of complexes such as thisRNA-cholesterol complex, by suppressing the expression of the RGM249gene (gene 2) over a long time, suppress the expression of thetelomerase reverse transcriptase (hTERT) gene. Additionally, if thecomplex is a complex that can be obtained by linking an uptake promoter(inducer) to the various types of RNA, the uptake efficiency into cellswill also improve. Therefore, these various types of RNA suppresstelomerase activity over a long time, and as a result, can suppresscarcinogenesis over a long period of time.

Because of this, medicines containing the abovementioned various typesof RNA such as siRNA and shRNA, or the abovementioned RNA-cholesterolcomplexes, suppress the expression of the telomerase reversetranscriptase (hTERT) gene when administered to the human body. Whereby,these medicines suppress telomerase activity, and as a result, canoptimally be used as cancer treatment agents that can suppresscarcinogenesis. Particularly, medicines containing the abovementionedRNA-cholesterol complexes have the advantage of making the interval ofadministration to the human body longer, because they suppresscarcinogenesis over a long period, due to the abovementioned actionmechanism.

The modes of embodiment of the present invention have been stated withreference to drawings, but these are examples of the present invention,and various constitutions other than the abovementioned ones can beutilized.

For example, in the abovementioned mode of embodiment, an siRNA is usedthat has, as a sense chain, a sequence that is a portion of the basesequence of the RGM376 gene (gene 1) or the RGM249 gene (gene 2), and ispredicted by BLOCK-iT RNAi Designer, but other RNAi design software mayalso be used, or RNA may be generated randomly and an siRNA that causesRNAi activity can be selected by experiment. An siRNA that suppressesthe expression of the RGM376 gene (gene 1) or the RGM249 gene (gene 2)can also be obtained in this way.

Herebelow, the present invention shall be further explained usingembodiments, but the present invention is not restricted to these.

<Investigation of miRNA Generation>

FIG. 11 is an electrophoresis diagram for explaining the cleaving of twotypes of mRNA with Dicer. Prior to conducting the below-describedembodiments, we conducted an investigation of the generation of miRNAfrom the RGM376 gene (gene 1) and the RGM249 gene (gene 2).

Here, the method of experiment was conducted as below. That is, theRGM249 and RGM376 genes were implanted into expression vectors having T7promoters inside, and using a mass RNA regulation method using T7 RNApolymerase (T7 RiboMAX (registered trademark) Express Large Scale RNAProduction System: Promega), RNA of both RGM249 (gene 1 mRNA) and RGM376(gene 2 mRNA) were generated, and were cleaved with an Rnase III familyDicer (Turbo Dicer: Genlantis). As a result, RNA corresponding to Dicedgene 1 and Diced gene 2 were obtained. Thereafter, the reaction productswere electrophoresed with a 30% acrylamide gel and visualized.

When mRNA of the RGM376 gene (gene 1) and the RGM249 gene (gene 2) werecleaved with Dicer, bands each having a size of approximately 25 bp weregenerated. The existence of these bands with a size of approximately 25bp suggested that by cleaving the RGM376 gene (gene 1) and the RGM249gene (gene 2) inside cells, miRNA was being generated.

Embodiment 1

In the present embodiment, a functional analysis was carried out of agene (RGM376 gene (gene 1)) that suppresses the expression of the mRNAof the telomerase reverse transcriptase gene hTERT.

More specifically, in the present embodiment, (1) as an investigation ofgene expression, comparison of a liver cancer cell line and aprimary-cultured hepatocyte cell line, and comparison of tissue from acancerous portion and a non-cancerous portion of liver was conducted,and (2) as a transfection of a gene by a forced expression vector, ananalysis of a growth curve and telomerase activity was conducted.

Further, when conducting the abovementioned experiments, in order toensure that a mutation is not occurring during the process of PCR, thesequence of PCR products was confirmed.

FIG. 12 is an electrophoresis diagram showing the differentiationspecific expression of RGM376 in liver cancer cells. Using Alexandercells (undifferentiated), HepG2 (high differentiation), HLF(undifferentiated), HuH7 (high differentiation), HMc-Li7 (high to mediumdifferentiation), and primary cultured hepatocytes, and RT-PCR andelectrophoresis, the level of expression of the mRNA of the RGM376 gene,the hTERT gene, and the β-actin gene inside cells was examined.

Here, the experiment was done in the following manner. That is, theexpression of the RGM249, the hTERT, and the β-actin gene in 5 types ofliver cancer cell lines was detected by using the RT-PCR method,reacting for 30-35 cycles, and conducting electrophoresis. Primarycultured hepatocytes (KIA) were reacted under the same conditions as theliver cancer cell lines, and the expression of each gene was detected.As the detection method, electrophoresis was done with a 1% agarose gel.

The results of the electrophoresis were that the expression of the hTERTgene increased, and the expression of the RGM376 gene decreased. On theother hand, in normal cells, the expression of the hTERT gene decreased,and the expression of the RGM376 gene increased. That is, in livercancer cell lines, differentiation specific expression was recognized.If the differentiation specific expression results in tissue fromcancerous portions and noncancerous portions of liver obtained in FIG.12, and the unshown clinical results are summarized, they are asfollows.

(1) Among tissue from noncancerous portions, the expression of theRGM376 gene was recognized in 84.4% (38 of 45 samples, unshown portionsincluded).

(2) In comparisons with clinical parameters (not shown), the expressionof the RGM376 gene in cancerous portions was significant according tot-tests for tumor size (P=0.003), tumor number (P=0.047), level ofdifferentiation (P=0.021), and vascular infiltration (P<0.001).

(3) In comparisons in terms of recurrence prediction factors (notshown), a strong correlation with capsule infiltration (P=0.001) wasseen.

If all of these results are considered in toto, it can be presumed thatwhen the strength of expression of the RGM376 gene is low, the level ofdifferentiation of cancer cells becomes high, and capsule infiltrationbecomes low. On the other hand, if the strength of expression of theRGM376 gene is high, the level of differentiation of cancer cellsbecomes approximately medium, and the capsule infiltration becomes high.

FIG. 13 is a graph and an electrophoresis diagram for explaining thesuppression of telomerase activity due to the overexpression of RGM376.In FIG. 13( a), the horizontal axis shows the number of days ofculturing after selection, and the vertical axis shows the number ofcells. Further, HM indicates the HMc-Li7 line (bought from Clontech),THM indicates HM telomerized by pLXIN-hTERT, and VHM indicates HM withonly pLXIN (obtained through the courtesy of Dr. Tahara of HiroshimaUniversity Department of Medicine) transfected. HM+RGM376 indicates genetransfected cell lines wherein RGM376 is transfected into HMc-Li7 celllines.

Here, the method of experiment was carried out as follows. That is,regarding liver cancer cell line HM, cell lines where RGM376 wastransfected into HM cell lines, THM cell lines (provided by HisatoshiTahara of Hiroshima University) wherein hTERT is forcibly expressed inHM cell lines, and VHM cell lines wherein only the vector istransfected, were used. Further, RGM376 was transfected by implantinginto the expression vector pEGFP-C1 (Clontech at the time).Additionally, the telomerase activity in cells wherein RGM376 and thelike are transfected was detected using the TRAPeze detection kit (OncorInc.). During this detection, the detection was done by using a reagentprovided by Oncor and following the indicated protocol.

As shown in the graph of FIG. 13( a), when the RGM376 gene isoverexpressed in HMc-Li7 cell lines, cells that were originally in agrowth phase entered a growth suppression phase after approximately 25days, and the number of cells started decreasing after approximately 50days. On the other hand, when the RGM376 gene is not overexpressed inHMc-Li7 cell lines, the growth phase continues undisturbed.Additionally, if the RGM376 gene and the hTERT gene are overexpressedtogether in HMc-Li7 cell lines, the growth phase continues undisturbed.On the other hand, in revertant cell lines (Revertant), the growthsuppression phase was entered approximately 25 days later, and thenumber of cells started increasing after approximately 50 days.

FIG. 13( b) shows the results of having done RT-PCR and electrophoresisin each of the THM, THM+RGM376, HM, HM+RGM376, VHM, and Revertant celllines, for both the cases where Rnase treatment was carried out and notcarried out. Further, for the HM+RGM376 and Revertant cell lines,samples were taken on the day marked with a black circle in FIG. 13( a).

FIG. 14 shows an electrophoresis diagram of the expression of hTERT andRGM376 in transformed cells. For cells in a growth period (in growth), asuppression period (in suppression), and revertants (in revertant), theamount of expression of mRNA of the hTERT gene, the RGM376 gene, and theβ-actin gene were examined.

At this time, the method of experimentation was as follows. That is, incells genetically transfected with RGM376 in FIG. 13, the expression ofeach gene was examined by RT-PCR in the growth phase, suppression phase,and revertant phase due to the beginning of the dropping out of thetransfected gene, using RNA treated by Dnase. For the RNA purification,the SV total RNA isolation system of Promega was used.

As a result, in the growth phase, the amount of expression of the hTERTgene was low, while the amount of expression of the RGM376 gene washigh. Additionally, during the suppression phase, the amount ofexpression of the hTERT gene was low, while the amount of expression ofthe RGM376 gene was high. However, in the suppression phase, incomparison with the growth phase, the amount of expression of the hTERTgene was high, while the amount of expression of the RGM376 gene waslow. On the other hand, in revertant cell lines, the amount ofexpression of the hTERT gene was high, while the amount of expression ofthe RGM376 gene was low.

From these results, regarding gene expression, it can be seen that inhighly differentiated cancer in comparison with undifferentiated cancer,the RGM376 is highly expressed. Additionally, it can be seen that theamount of expression of the RGM376 gene is strongly correlated toclinical parameters that relate to the degree of malignancy of cancer(tumor size, tumor number, differentiation, vascular infiltration).Whereby, it is suggested that the expression of the RGM376 gene isrelated to the degree of differentiation of tumors. That is, it issuggested that the RGM376 gene has a function of suppressing telomeraseactivity within cells, and suppressing the canceration of cells.

Additionally, it is suggested that the overexpression of the RGM376 genehas a function of suppressing the growth of cells that have passed thequiescent phase, and aging cells, and promoting cell death.Additionally, it is suggested that the overexpression of the RGM376 genehas a function of decreasing telomerase activity. Further, it issuggested that the overexpression of the RGM376 gene has a function ofdecreasing the amount of expression of mRNA of the hTERT gene. That is,it can be presumed that the RGM376 gene is a gene that has atelomerase-mediated (hTERT) cancer suppressing activity.

FIG. 15 is an electrophoresis diagram showing the expression of genes inRGM376 siRNA transfected cells. Cells not transfected with siRNA werecontrols, and as an siRNA against the RGM376 gene, the siRNA comprisingthe abovementioned A and B sequences was used. RT-PCR andelectrophoresis was done on cells wherein each of these siRNA wastransfected, for the RGM376 gene, the hTERT gene and the β-actin gene.Additionally, the case where A and B were mixed and transfected was alsoexamined.

At this time, the method of experimentation was as follows. That is, theRGM376 siRNA was designed using the Block-it RNAi designer (homepage) ofInvitrogen Corporation, and the two types of siRNA synthesized by saidcompany, A and B, were genetically transfected using Lipofectamine 2000,into the liver cancer cell line HLF at a transfection concentration of25 nM. Further, a mixture of 25 nM each of A and B was also transfected.Further, regarding the expression of each gene, after RNA was purifiedfrom the transfected cells by the same method as FIG. 14, detection wasdone by the RT-PCR method.

FIG. 16 is an electrophoresis diagram and graph summarizing the hTERTsuppression results due to RGM376 siRNA. Additionally, FIG. 16 alsoshows the sequence of the sense chain of the siRNA (A and B) used inthis experiment.

At this time, the method of experimentation was as follows. That is, thetransfection of siRNA shown in FIG. 15 was attempted 3 times, the degreeof suppression of expression of RGM376 was examined by RT-PCR, and atthe same time, the strength of expression was measured using adensitometer on an electrophoresis image, and this is shown in a bargraph standardized by the measured values for no siRNA.

As shown in FIG. 16, if the expression of the RGM376 gene is suppressedby RGM376 siRNA, the expression of the hTERT gene is also similarlysuppressed. Among the RGM376 siRNA, the suppression effect is best whenA alone is used. Additionally, among the RGM376 siRNA, even when B aloneis used, an excellent suppression effect was obtained, but thesuppression effect was lower than for A. Further, among the RGM376siRNA, when both A and B were transfected, an excellent suppressioneffect was obtained, but the suppression effect was lower than for A.

Accordingly, if the expression of the RGM376 gene is suppressed byRGM376 siRNA, the expression of the hTERT gene is also similarlysuppressed. Whereby, telomerase activity can be suppressed by RGM376siRNA, and as a result, the canceration of cells can be suppressed.

Embodiment 2

In the present embodiment, a functional analysis was carried out of agene (RGM249 gene (gene 2)) that is expressed synchronously withtelomerase reverse transcriptase gene hTERT mRNA.

In more detail, (1) expression in liver cancer cell lines was examinedwith RT-PCR, (2) expression in liver tissue was examined with RT-PCR,(3) the relation to genes that relate to telomerase formation wasexamined, and (4) dsRNA derived from genes whose involvement issuggested was expressed, and transfection with siRNA was done, andobservations with RT-PCR, Cell proliferation assay, MTT assay, andmorphological changes were carried out.

Further, when performing the abovementioned experiments, in order toensure that no mutations have occurred in the process of PCT, thesequence of PCR products were confirmed.

FIG. 17 is an electrophoresis diagram showing differentiation specificexpression of RGM249 in liver cancer cells. Using Alexander cells(undifferentiated), HepG2 (high differentiation), HLF(undifferentiated), HuH7 (high differentiation), HMc-Li7 (T, N, C), andprimary cultured hepatocytes as samples, the level of expression of themRNA of the RGM376 gene, the hTERT gene, and the β-actin gene insidecells was examined by RT-PCR and electrophoresis.

Here, the experiment was done in the following manner. That is, by thesame method as in FIG. 12, the expression of RGM249, hTERT, RPL22, andβ-actin was examined by the RT-PCR method. Further, in FIG. 17, forHMc-Li7, T indicates total RNA, N indicates RNA extracted from nucleus,and C indicates RNA inside cells.

As a result of electrophoresis, for the RGM249 gene, expression in livercancer cell lines and primary cultured hepatocyte cell lines expressingthe hTERT gene was seen. Additionally, in cancer cells, the expressionof the hTERT gene increased, and the expression of the RGM249 genesynchronously increased. On the other hand, in normal cells, theexpression of hTERT cells decreased. That is, differentiation-specificexpression was seen in liver cancer cell lines.

FIG. 18 is an electrophoresis diagram showing the expression of genesdue to the transfection of RGM249 dsRNA into HMc-Li7. dsRNA designed tosuppress the expression of RGM249 was transfected into HMc-Li7 cells,and RT-PCR and electrophoresis was performed on each of the hTERT, hTR,TEP1, dyskerin, RPL22, Nola3, and RGM249 genes.

Here, the method of experimentation was as follows. That is, RGM249dsRNA was implanted into the restriction enzyme portion of a vector,Litmas 28i (New England BioLab), having a T7 promoter in dualdirections, and a double stranded RNA was generated with T7 RNApolymerase. After this double stranded RNA was purified, usingtransformed cells wherein Lipofectamine Plus is transfected into HM, thestrength of expression of the telomerase related genes (hTERT, hTR,TEP1, Dyskerin, RPL22, Nola3, RGM249) was measured by a densitometer,and the results shown in FIG. 18 were obtained.

As a result thereof, in HMc-Li7 cells wherein the expression of theRGM249 gene was suppressed, the expression of the hTERT gene and theRPL22 gene were also synchronously suppressed. Whereby, it is suggestedthat the RGM249 gene has a function of promoting the expression of thehTERT gene and the RPL22 gene.

FIG. 19 is an electrophoresis diagram showing the expression of a genedue to transfection of RPL22 dsRNA into HM. dsRNA designed to suppressthe expression of RPL22 was transfected into HMc-Li7 cells, and RT-PCRand electrophoresis was performed for each of the hTERT, TEP1, hTR,RGM249, dyskerin, Nola3, RPL22, and GAPDH genes.

At this time, the experimental method was as follows. That is, RPL22cDNA (TOYOBO) was purchased, and after culturing in an E. coli strain,plasmid DNA was purified, and the RPL22 cDNA was implanted into therestriction enzyme portion of Litamas 28i, and dsRNA of RPL22 wasobtained similarly to FIG. 18, and was transfected into HM usingLipofectamine Plus (Invitrogen). The abovementioned dsRNA wastransfected into transformed cells obtained in this way, at transfectionconcentrations of 40, 20, 10, and 0 μg/ml. After this, for each gene,expression was examined using RT-PCR, and electrophoresis was performed.

As a result, in HMc-Li7 cells wherein the expression of the RPL22 geneis suppressed, the expression of (1) the RGM249 gene, (2) the hTERTgene, and (3) dyskerin were also synchronously suppressed. Whereby, itis suggested that the RPL22 gene has a function of promoting theexpression of the RGM249 gene, the hTERT gene, and the dyskerin gene.

FIG. 20 is an electrophoresis diagram showing the expression of genesdue to the transfection of RGM249 specific siRNA into HLF. A specificsiRNA designed to suppress the expression of RGM249 (siRNA of theabovementioned (1) (indicated in the figure with a circled number) and(2) (indicated in the figure with a circled number)), and RT-PCR andelectrophoresis was done for each of the RGM249, hTERT, RPL22, and theβ-actin genes. As a result thereof, in HLF cells wherein the expressionof the RGM249 gene was suppressed by siRNA (1), the expression of thehTERT gene and the RPL22 gene were also synchronously suppressed.

At this time, the method of experimentation was as follows. That is,similarly with FIG. 15, the two types of RGM249 siRNA (1) and (2),designed and synthesized by Block-it RNAi designer, was transfected intothe liver cancer cell line HLF using Lipofectamine 2000 (Invitrogen),RNA was extracted from the transfected cells, RT-PCR was performed, andthe strength of expression was visualized by electrophoresis anddetected.

FIG. 21 is a graph summarizing the expression suppression rate of themRNA of each gene after transfection with RGM249 specific siRNA. Asshown in FIG. 21, if the expression of the RGM249 gene is suppressed byRGM249 specific siRNA (1) (indicated in the figure with a circlednumber), the expression of the hTERT gene and the RPL22 gene are alsosimilarly suppressed. Additionally, among the RGM249 specific siRNA,when (2) (indicated in the figure by a circled number) was used alone,and when (1) and (2) were both transfected, the expression suppressioneffect on the hTERT gene and the RPL22 gene was lower than when (1) wasused alone.

Therefore, when the expression of the RGM249 gene is suppressed byRGM249 specific siRNA, the expression of the hTERT gene and the RPL22gene are also suppressed. Whereby, telomerase activity can be suppressedby RGM249 specific siRNA (1), and as a result thereof, the cancerationof cells can be suppressed.

At this time, the method of experimentation was as follows. That is, thetransfection experiment examined in FIG. 20 was performed 3 times, theelectrophoresis image was image scanned by a densitometer, the measuredvalues were standardized relative to no siRNA, and the degree ofsuppression of genes due to siRNA was shown with a bar graph.

From these experimental results regarding the RGM249 gene, it was foundthat the RGM249 gene was expressed in approximately 80% of liver cancercell lines and liver cancer tissue. Additionally, due to RGM249 specificsiRNA genetic transfection experiments, it was found that siRNA derivedfrom RGM249 suppress the expression of the hTERT gene and the RPL22gene. Further, although not shown in a diagram, when liver tissue wasexamined, it was found that among the correlations between the variousclinical parameters, there was a significant correlation betweenPIVKA-2, which is a tumor marker for liver cancer, and the amount ofexpression of the RGM249 gene, by t-test, multivariable analysis, andPearson's correlation test.

Whereby, it is presumed that in liver cancer cells expressing the hTERTgene, primary cultured liver cells, liver cancer tissue, and non-livercancer tissue, the RGM249 gene is uniformly expressed. Additionally,there is a possibility that the RGM249 gene is involved in theexpression of the hTERT gene and the RPL22 gene. Further, theinvolvement of the RGM249 gene in carcinogenesis is suggested.

<Relation Between RGM376 and RGM249>

FIG. 22 is an electrophoresis diagram showing the expression of RGM376due to the transfection of RGM249 siRNA, and the expression of RGM249due to the transfection of RGM376 siRNA.

At this time, the method of experimentation is as follows. That is, inthe transfection experiments performed in FIG. 21 and FIG. 15, (a) forRGM249 transfection, the expression of RGM376, and (b) for RGM376transfection, the expression of RGM249, was detected by extracting RNAfrom the transfected cells and performing RT-PCR.

FIG. 22( a) are the results of having observed the influence oftransfection with RGM249 siRNA ((1) (indicated within the diagram with acircled number) and (2) (indicated within the diagram with a circlednumber)), by RT-PCR and electrophoresis. When the expression of theRGM249 gene is suppressed by RGM249 specific siRNA (1), the expressionof the RGM376 gene is similarly suppressed. Additionally, among the RGMspecific 249 siRNA, when (2) is used alone, and when (1) and (2) areboth transfected, the expression suppression effect of the RGM376 genewas lower than when (1) was used alone.

FIG. 22( b) are the results of having observed the influence oftransfection with RGM376 siRNA (A and B) on the expression of RGM249, byRT-PCR and electrophoresis. When the expression of the RGM376 gene wassuppressed by RGM376 specific siRNA, the expression of the RGM249 genewas not suppressed.

From the abovementioned results, it can be seen that the RGM249 genecontrols the expression of the RGM376 gene. When doing so, it ispresumed that the RGM249 gene controls the expression of the RGM376 genein the form of promotion. On the other hand, it can be seen that theRGM376 gene does not control the expression of the RGM249 gene.

FIG. 24 is a conceptual diagram for explaining the mechanism wherebyshRNA is generated from plasmids whose purpose is to generate shRNA, andthe expression of a target gene is suppressed. As shown in the lefthandside of FIG. 24, a plasmid vector was constructed containing a DNAsequence complementary to the region wherein the RNA sequence comprisingCGAA is disposed between the sense strand and the antisense strand ofthe abovementioned RGM249 siRNA. Further, this DNA sequence was placeddownstream of the promoter of the plasmid vector.

FIG. 28 is a figure for explaining the RGM249 shRNA sequence designedbased upon gene 2. As shown in the figure, by annealing a singlestranded DNA comprising the DNA sequence shown in SEQ ID No. 33, and asingle stranded DNA comprising the DNA sequence shown in SEQ ID No. 34,a double stranded DNA wherein these two single stranded DNA arecomplementarily linked was obtained. Then, a vector for expressing theRGM249 shRNA was produced by implanting this double stranded DNA into aplasmid vector as shown above.

Next, from the DNA sequence of this plasmid vector, an RNA strandwherein an RNA sequence comprising CGAA is disposed between the sensestrand and the antisense strand of the abovementioned RGM249 siRNA, aloop is formed in the CGAA portion, and an RGM249 shRNA was generated byforming a stem by linking the sense strand and the antisense strand.

On the other hand, as shown in the righthand side of FIG. 24, an mtplasmid vector was constructed containing a DNA sequence complementaryto the region wherein an RNA sequence comprising CGAA is disposedbetween the sense strand of the abovementioned RGM249 siRNA and anantisense variant strand wherein one of the 8th T residues from the 5′end of the antisense strand is deleted. This DNA sequence was placeddownstream of the promoter of the mt plasmid vector.

FIG. 29 is a diagram for explaining the sequence of RGM249 variant shRNAdesigned based upon gene 2. As shown in this diagram, by annealing asingle stranded DNA comprising the DNA sequence shown in SEQ ID No. 35,wherein one of the 8th T residues from the 5′ end of the antisensetarget sequence of the single stranded DNA comprising the DNA sequenceshown in SEQ ID No. 33 is deleted, and a single stranded DNA comprisingthe DNA sequence shown in SEQ ID No. 36, a double stranded DNA linkingthese two single stranded DNA complementarily was obtained. Then, avector for expressing RGM249 variant shRNA was produced by implantingthis double stranded DNA into a plasmid vector as described above.

Then, from the DNA sequence of this mt plasmid vector, an RGM249 variantshRNA made by disposing an RNA sequence comprising CGAA between thesense strand and an antisense variant strand of the RGM249 siRNAdescribed above, forming a loop at the CGAA portion, and then forming astem by linking together the sense strand and the antisense strand, wasgenerated. Then, the expression of the RGM249 gene was suppressed bythis RGM249 variant shRNA.

FIG. 25 is an electrophoresis diagram showing the expression of mRNA ofeach gene after transfection with RGM249 shRNA. Plasmid vectors and mtplasmid vectors that generate specific shRNA and variant shRNA designedto suppress the expression of RGM249 as described above were transfectedinto HLF cells, and RT-PCR and electrophoresis was performed for each ofthe RGM249, hTERT, RPL22, and the β-actin genes. As a result thereof, inHLF cells wherein the expression of the RGM249 gene was suppressed byshRNA and variant shRNA, the expression of the hTERT gene and the RPL22gene was also synchronously suppressed.

At this time, the experimental method was as follows. That is, similarlyto FIG. 20, plasmid vectors and mt plasmid vectors that generatespecific shRNA and variant shRNA designed to suppress the expression ofRGM249, described above, designed and synthesized with Block-it RNAidesigner, was transfected into the liver cancer cell line HLF, RNA wasextracted from transfected cells expressing the shRNA and variant shRNA,RT-PCR was performed, the strength of expression was visualized byelectrophoresis, and detected.

FIG. 26 is a graph summarizing the expression suppression rate of mRNAof each gene after transfection with RGM249 shRNA. As shown in FIG. 26,when the expression of the RGM249 gene is suppressed by plasmid vectorsand mt plasmid vectors that generate specific shRNA and variant shRNAdesigned to suppress the expression of RGM249, described above, theexpression of the hTERT gene and the RPL22 gene are also similarlysuppressed. Additionally, in cases where one of either the plasmidvectors or the mt plasmid vectors is used, the expression suppressioneffect against the hTERT gene and the RPL22 gene was approximately thesame.

Therefore, if the expression of the RGM249 gene is suppressed byspecific shRNA and variant shRNA designed so as to suppress theexpression of RGM249, the hTERT gene and the RPL22 gene are alsosimilarly suppressed. Whereby, telomerase activity can be suppressed byspecific shRNA and variant shRNA designed so as to suppress theexpression of RGM249, and as a result thereof, the canceration of cellscan be suppressed.

At this time, the experimental method was as follows. That is, thetransfection experiment considered in FIG. 25 was performed three times,the electrophoresis image was image scanned using a densitometer, themeasured values were standardized relative to no siRNA, and the degreeof suppression of the siRNA gene was shown in a bar graph.

From these experimental results regarding the RGM249 gene, it was foundfrom experiments of the genetic transfection of specific shRNA andvariant shRNA designed so as to suppress the expression of RGM249, thatshRNA and variant shRNA derived from RGM249 suppress the expression ofthe hTERT gene and the RPL22 gene. Whereby, there is the possibilitythat the RGM249 gene is involved in the expression of the hTERT gene andthe RPL22 gene.

The present invention has now been explained based upon embodiments.These embodiments are merely examples, and it may be understood by thoseskilled in the art that various variant examples are possible, and suchvariant examples are also within the scope of the present invention.

For example, in the abovementioned embodiment, as an siRNA, Stealth(registered trademark) RNAi from Invitrogen Corporation was used, butsiRNA or shRNA from other manufacturers, having differing structures,may be used. This is because in such cases also, as long as theexpression of the target mRNA can be suppressed, a similar effect can beobtained.

For example, in the abovementioned embodiments, siRNA, or siRNA (miRNA)obtained by cleaving siRNA with Dicer was explained, but this is notparticularly restricted. For example, in vivo, a mechanism is alsopresumed where, from a precursor miRNA gene, pri-miRNA of less thanapproximately 70 bp is generated by Drosha, and becomes pre-miRNA by wayof Dicer (see conceptual diagram for explaining the presumed mechanismfor translation silencing due to the Micro RNAs of FIG. 23). A similareffect to that of the abovementioned embodiment can be achieved also bya mechanism whereby miRNA obtained through this pri-miRNA and pre-miRNAbecomes miRNA and suppresses the translation of the target mRNA withincells.

In the abovementioned embodiments, there were explanations involvingRNA, but the RNA can also function as RNP. An RNP (ribonucleoprotein) isa complex that functions by RNA linking to a protein. A mechanismwhereby a target is cleaved by forming RNP by these tiny RNA beingimplanted into RISC can be presumed.

INDUSTRIAL APPLICABILITY

As shown above, since the hTERT expression regulatory gene according tothe present invention has the effect of regulating the expression of thetelomerase reverse transcriptase gene in mammalian cells, this gene, orspecific RNA that suppresses the expression of this gene is useful as aresearch tool, as a raw material for medicines (cancer treatment agent),and the like.

1. An hTERT expression regulatory gene comprising the base sequence shown in SEQ ID No.
 2. 2. An isolated hTERT expression regulatory gene consisting of a base sequence wherein one base is deleted from, replaced in, or added to the base sequence shown in SEQ ID No.
 2. 3. An hTERT regulatory gene expression vector constructed so as to allow expression, by linking an isolated hTERT expression regulatory gene recited in claim 2 to a vector.
 4. An isolated hTERT expression regulatory RNA, being a transcription product of an isolated hTERT regulatory gene recited in claim
 2. 5. An isolated hTERT expression regulatory RNA comprising the base sequence shown in SEQ ID No.
 4. 6. An isolated hTERT expression regulatory RNA consisting of a base sequence wherein one base is deleted from, replaced in, or added to the base sequence shown in SEQ ID No.
 4. 7. An hTERT regulatory gene expression vector constructed so as to allow expression, by linking an hTERT expression regulatory gene recited in claim 1 to a vector.
 8. An isolated hTERT expression regulatory RNA, being a transcription product of an isolated hTERT regulatory gene recited in claim
 1. 